Since the development of the Polymerase Chain Reaction (PCR), the demand for fast, reliable, and cost-effective tests for the detection of specific nucleic acid sequences has led to the development of a variety of new assay techniques. One of these, referred to as the INVADER™ (a trademark of Third Wave Technologies, Madison, Wis.) Assay, Invasion Cleavage Assay, or Invasion Assay, does not require the use of PCR. Invasion assays are highly sensitive and can be used to determine, for example, single-base differences of specific nucleotide targets. See, e.g., Lyamichev, V., et al., Nature Biotech. 17:292-296 (1999); and Ryan, D., et al., Molecular Diag. 4(2):135-144 (1999), each of which is incorporated herein by reference.
Invasion assays are based on the ability of some enzymes (e.g., endonucleases) to cleave nucleotide “flaps,” such as those shown in FIG. 1A, that are formed when an oligonucleotide complementary to part of a DNA template overlaps with another oligonucleotide complementary to another part of the template. Specific enzymes, such as FEN 1, cleave flaps formed when the 3′ end of an upstream oligonucleotide complementary to part of a DNA template overlaps with the 5′ end of a downstream oligonucleotide complementary to another part of the DNA template. As shown in FIG. 1B, cleavage by such an enzyme of a downstream oligonucleotide (identified as Probe in FIG. 1B) provides a fragment that corresponds to the overlap between the two oligonucleotides complementary to the template. See Lyamichev, V., et al., Nature Biotech. 17:292-296 (1999).
Many endonucleases are thermostable, and can be used near the melt temperatures of the probe oligonucleotides they cleave. See Kaiser, M. W., et al., J. Biol. Chem. 274(30):21387-21394 (1999), and patents and published patent applications referred to herein. Most invasion assays take advantage of this characteristic by using a molar excess of a probe oligonucleotide that, at or near its melt temperature, will rapidly associate and disassociate with the template DNA. This allows a new, uncleaved probe to replace a cleaved probe such that when the experiment is complete, the quantity of cleaved fluorescent fragments is significantly larger than the amount of template DNA. See, Lyamichev, V., et al., Nature Biotech. 17:292-296 (1999).
FIG. 2 provides a representation of an invasion assay that is disclosed by, for example, U.S. Pat. No. 5,994,069, which is incorporated herein by reference. In this assay, the fragment cleaved from a probe oligonucleotide invades a second structure (referred to herein as a “reporter precursor”) that contains fluorescent reporter R and a quencher Q. Association of the probe fragment with the second structure forms a flap that is cleaved by enzyme in the mixture. This cleavage breaks the connection between the reporter R and quencher Q and provides a fluorescent reporter fragment.
Theoretically, cleavage of the reporter precursor shown in FIG. 2 can occur only after a secondary invader oligonucleotide fragment is created by the primary invasion reaction. In some cases, however, the primary invader oligonucleotide, which is intended to only bind to the template DNA, also binds to some degree to the secondary structure, causing cleavage of that structure. This cleavage creates a background signal that must be considered when determining the result of the assay. Consequently, invasion assays such as are represented by FIG. 2 have been run with a control, i.e., an assay in which template, or target, DNA is not present. The fluorescent signal measured at the completion of this control assay is the signal due to the undesired cleavage of the secondary structure by the primary invader oligonucleotide. This control signal is subtracted from that measured at the completion of the test assay (i.e., the assay with the template DNA) to provide an approximation of the signal due only to an overlap between the invader oligonucleotide and probe oligonucleotides in the primary invasion reaction. In theory, a positive net signal indicates that the invader oligonucleotide and probe oligonucleotide are complementary to different parts of the DNA template and overlap.
The invasion assay is a powerful analytical tool that can be used to supplement, or even replace, existing assays such as PCR. It is desirable, however, to increase its speed and accuracy. It is also desirable to reduce the amount of probe material that must be used to provide a useful (e.g., reliable) result. These and other needs are met by the invention disclosed herein.